In 33 healthy dogs, 66 bronchoalveolar lavage samples from the right and left caudal lung lobes were analyzed for volume of return, cellularity, differential cellularity, and immunophenotypic lymphocyte subpopulations. Lavage return was 64.8% (mean) following 3 sequential 25-ml lavages, for a total lavage volume of 75 ml. With this technique, 21.1 X 10(6) cells/sample (mean) were obtained. The cellular components of bronchoalveolar lavage samples, in decreasing order of frequency, were alveolar macrophages (79.4%), lymphocytes (13.5%), eosinophils (3.6%), mast cells (2.1%), epithelial cells (0.8%), and neutrophils (0.6%). Mean alveolar lymphocyte subpopulation frequencies, determined in 18 samples, for pan T, CD4, and CD8 cells were 52, 21.9, and 17.8%, respectively, with a CD4/CD8 ratio of 1.3. Variables analyzed did not vary between right and left caudal lung lobes, nor were they affected by body weight.

Fifty serum samples from dogs with clinical signs of hypothyroidism and autoantibodies (AA) to thyroglobulin (Tg), thyroxine, or triiodothyronine were screened for AA to thyroid peroxidase (TPO). Thyroid peroxidase is the antigen against which microsomal AA are formed in human beings with lymphocytic thyroiditis. The TPO was isolated from canine thyroid tissue, using a modification of the procedure for purifying porcine TPO. The enzyme was solubilized from the membrane, using a deoxycholate-trypsin solution, followed by ammonium sulfate precipitation and diethylaminoethyl Sephadex chromatography. Activity of TPO was determined, using an iodide oxidation assay and a guaiacol assay. A monoclonal antibody to canine Tg, coupled to an immunoaffinity column, was used to eliminate the contaminating Tg from the TPO preparation. Using the TPO preparation as an antigen, an ELISA was performed on 10 serum samples and immunoblot assays were performed on 50 canine sera. Autoantibodies to TPO were not found in any of the sera. Assays also were performed, using purified porcine and human TPO and evidence of cross-reactivity with canine TPO was not identified. The absence of AA to TPO in dogs suggests a different pathogenesis for autoimmune thyroid disease in dogs than that hypothesized for lymphocytic thyroiditis in human beings.

Tumor necrosis factor-alpha (TNF) is an important mediator of endotoxin-induced pathologic changes. To help define the role of TNF in equids with endotoxemia, the effects of pretreatment with a murine monoclonal antibody (MAB) against equine TNF were evaluated in Miniature Horses given endotoxin. Five horses were given TNF MAB at a dosage of 1.86 mg/kg of body weight, IV, and 5 were given control MAB. Five minutes later, lipopolysaccharide (LPS; Escherichia coli O55:B5), 0.25 micrograms/kg, was given to all horses by bolus IV infusion. Clinical signs of disease were monitored at intervals up to 24 hours after LPS infusion, and blood was taken for determination of WBC count, PCV, plasma total protein concentration, plasma TNF activity, and serum MAB concentration. Reduction of plasma TNF activity in anti-TNF-treated horses was highly significant (P < 0.001), compared with that in control horses. Horses given TNF MAB had significantly improved clinical abnormality score (P < 0.010), lower heart rate (P < 0.001), and higher WBC count (P < 0.001), compared with horses given control MAB. Rectal temperature, respiratory rate, PCV, and plasma total protein concentration were not significantly different between groups. Serum MAB concentration peaked at 68 micrograms/ml 30 minutes after the end of antibody infusion in both groups. Neutralization of LPS-induced TNF activity reduced the hematologic and clinical responses of horses given LPS IV.

A murine monoclonal antibody (MAB) 6G7 generated against tachyzoites of Neospora caninum recognized 8 major and several minor antigens, as observed by western blot analysis. Relative rate of migration of the 8 major antigens ranged from 31 to 97.4 kd. In addition, MAB 6G7 recognized a Toxoplasma gondii tachyzoite antigen with a relative rate of migration of 107 kd. Immunogold labeling of N caninum tachyzoites grown in human foreskin fibroblast cells indicated that MAB 6G7 binds to micronemes, dense granules, basal portions of rhoptries, and intravacuolar tubules within the parasitophorous vacuole. Monoclonal antibody 6G7 also bound to micronemes and basal portions of rhoptries within tachyzoites of T gondii. Monoclonal antibody 6G7 did not significantly inhibit development of tachyzoites in vitro.

Serologic testing to detect persistent IgG titer directed at Salmonella lipopolysaccharide (LPS) has proven useful in detecting Salmonella carrier cattle without clinical signs of disease and in seroepidemiologic studies. Although little cross-reactivity exists between most Salmonella serogroups, groups B (O1, 4 [5], 12) and D (O1, 9, 12) share somatic (LPS cell wall) antigens O1 and O12, which results in some cross-reactions. This may be unimportant in most instances, because group-B and group-D carriers need to be identified and culled. It may be desirable in some situations, such as when trying to control S dublin, to determine which serogroup is present in a given herd. For this reason, a procedure to produce a pure O9 group-D antigen was developed. Salmonella dublin (group D) was grown by use of standard procedures, and LPS was extracted by use of the phenol-water method. The LPS was then oxidized with sodium periodate, dialyzed, reduced with sodium borohydride, cleaved with hydrochloric acid, and again dialyzed. This procedure successfully cleaved the saccharides comprising O antigens 1 and 12, leaving a pure O9 ELISA antigen. Sera from cattle vaccinated or naturally infected with S typhimurium, S agona, and S schwarzengrund (all group B), S montevideo (group C1), and S dublin (group D) were tested by ELISA, using modified and unmodified antigens. When the ELISA antigen used was the chemically modified (pure O9) group-D antigen, elimination of cross-reactions confirmed the structural loss of cross-reacting O1 and O12 antigens.

Binding of endogenous and exogenous homologous IgG, and IgM to bovine neutrophils before and after in vitro migration through micropore filters, and in vivo migration through mammary tissues after intramammary injection of endotoxin was evaluated by use of flow cytometry. Immunoglobulin binding to neutrophils at 4 and 37 C was also evaluated. Before and after in vitro migration, neutrophils with endogenously bound IgG, and IgM averaged 1 and 2% and 23 and 7%, respectively. Before and after in vivo migration, IgG2 and IgM binding averaged 1 and 7% and 26 and 15%, respectively. Before and after in vitro migration, binding of purified IgG2 and IgM averaged 75 and 67% and 8 and 24%, respectively. Before and after in vivo migration, percentage of neutrophils binding purified IgG2 and IgM averaged 92 and 98% and 54 and 70%, respectively. When serum was used as a source of exogenous immunoglobulins, binding of total Igg after in vitro migration increased from 5% to 28% and of IgM from 4% to 20%. After in vivo migration, binding increased from 21% to 47% and from 24% to 56%, respectively. Exogenous binding of IgG2 at 4 and 37 C averaged 75 and 84%, and binding of IgM averaged 8% at either temperature. Endogenous IgG2 was unaffected by temperature; however, binding of IgM decreased from 23% at 4 C to 2% at 37 C. These data indicate that endogenous binding was higher for IgM before migration than after migration, in vitro and in vivo. Furthermore, migration in vivo through cellular matrices induced receptor upregulation for IgG and IgM. Source and concentration of ligand and serum components, other than immunoglobulins, appeared to contribute to receptor expression and availability. Neutrophils that were exposed to endotoxin and migrated into milk expressed more receptors than did unstimulated and nonmigrating neutrophils. The association of IgM with its receptor was temperature-dependent.

An antigen extract of Dictyocaulus viviparus was analyzed by use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the antigen-recognition patterns of serum antibody from cattle not infected, cattle infected with D viviparus, and cattle with unknown history of D viviparus were analyzed by the use of ELISA and western blotting techniques. Cross-reactive antibody-recognition patterns were determined by comparing western blots of D viviparus-positive sera with blots of D viviparus-negative sera obtained from cattle singly infected with Bunostomum phlebotomum, Cooperia oncophora, C punctata, Nematodirus helvetianus, Oesophagostomum radiatum, or Ostertagia ostertagi. Five antigen bands unique to D viviparus were identified, and their frequency of appearance in western blots of sera from verified D viviparus-positive and -negative cattle, and sera from cattle exposed to the parasite, but with unknown D viviparus immune status, were determined. Of the 5 antigens unique to D viviparus, 29- and 19-kd bands had the highest frequencies of reaction (45.9 and 59.0%, respectively) with the test sera. These bands had strong reactivity with sera containing antibodies to D viviparus and did not react with the heterologous sera. We conclude that the 29- and 19-kd antigens may be useful for developing an improved serodiagnostic test for D viviparus infections in cattle.

An ELISA was developed to detect antibodies to the 41-kd flagellin (P41) of Borrelia burgdorferi in serum obtained from cattle. Absorption studies, immunoblot analysis, immunoelectron microscopy, and correlation of results of the P41-ELISA and the P39-ELISA as well as measurement of the antibody to P41 in calves challenge-exposed with Borrelia theileri were used to assess the specificity of the P41-ELISA. Antigens derived from Escherichia coli, Leptospira interrogans serovar hardjo, and B burgdorferi were used for absorption studies and immunoblot analysis. Antibodies to P41 of B burgdorferi cross-reacted with antigens of E coli, but were not cross-reactive with L hardjo. A value 3 SD higher than the mean of the negative-control population of cattle was defined as the minimum value cutoff value) for a positive result by the P41-ELISA. Use of this value for classification of test results reduced the predicted rate of false-positive results attributable to E coli cross-reactivity to 1%. Immunoblot analysis revealed that test-positive serum from cattle reacted mainly with 41-, 39-, 34-, and 31-kd proteins of B burgdorferi, as well as several smaller proteins. Immunoelectron microscopy revealed that serum from cattle that was test-positive by the P41-ELISA bound to the flagellin and outer membrane of B burgdorferi. Results of absorption studies, immunoblot analysis, and immunoelectron microscopy were correlated and indicated that serum from cattle that was test-positive by P41-ELISA had stronger reactivity to B burgdorferi antigens than to antigens of E coli or L hardjo. The concentrations of antibodies measured by P41-ELISA and P39-ELISA testing were highly correlated [R(2)=0.78]. Calves challenge-exposed with B theileri also had test-positive results by the P-41-ELISA as early as 2 weeks after exposure, but serum antibody concentrations decreased to prechallenge-exposure concentrations by 9 weeks after exposure. We concluded that the P41-ELISA was useful as a screening method to detect B burgdorferi infections in cattle.

A method for quantification of serum total IgE concentration in dogs by use of an ELISA containing monoclonal mouse anti-canine IgE was developed. Microtitration plates were coated with monoclonal mouse anti-canine IgE. Test sera and reference serum dilutions were added, followed by biotinylated monoclonal mouse anti-canine IgE. Avidin-alkaline phosphatase conjugate was added, and color development was measured spectrophotometrically, using a microtitration plate reader. Quantitative results were obtained by assigning to a reference serum a value of 100 IgE units/ml. Absorbance values of unknown samples were converted into IgE units by comparison with a standard curve generated by measurement of reference serum dilutions. Intra- and interassay coefficients of variation were 5 and 7%, respectively, and assay sensitivity was 1 U/ml. The assay was used to establish a normal range for total IgE concentrations in 30 healthy dogs. Total IgE concentration in healthy dogs followed a skewed distribution and ranged from < 1 to 91.2 U/ml, with a geometric mean value of 7.1 U/ml. The IgE concentration was remarkably stable in serum samples subjected to 25 freeze/ thaw cycles or incubation at approximately 25 C (room temperature) for up to 10 days. Comparison of total IgE concentrations in 23 serum samples assayed by use of double-overlay radial immunodiffusion and ELISA yielded correlation coefficient of 0.94. Comparison of the reference serum standard curve with serial dilutions of a purified IgE solution of known concentration yielded a range of values for the IgE unit of 0.7 to 2.0 micrograms.

Polymyxin B and an antiserum against an Re mutant Salmonella typhimurium were evaluated for protective effect in an equine model of endotoxemia. Six 3- to 5-month-old foals were given endotoxin (0.25 micrograms/kg of body weight) IV after no pretreatment, or pretreatment with polymyxin B (6,000 U/kg, IV) or S typhimurium antiserum (1.5 ml/kg, IV). When given without pretreatment, endotoxin caused transient recumbency and increases in rectal temperature, and heart and respiratory rates. In addition, leukopenia and increases in circulating tumor necrosis factor (TNF) and interleukin 6 (IL-6) activities were detected. Compared with results obtained when endotoxin was given alone, pretreatment with polymyxin B resulted in significantly (P < 0.05) lower maximal plasma TNF and IL-6 activities, and significantly lower rectal temperature and respiratory rate. In contrast, compared with effects of endotoxin given without pretreatment, use of antiserum was associated with significantly (P < 0.05) higher respiratory rate, maximal plasma IL-6 activity, and total TNF response (as determined by areas under curves of plasma TNF vs time). These results indicate that polymyxin B may have potential as a treatment for equine endotoxemia. Salmonella typhimurium antiserum had no positive effect in this model, and, under certain conditions, may exacerbate the actions of endotoxin.